TX 560 
.B5 W3 
Copy 1 

Ube Tllnivetsit^ of dbicaao 



THE CHEMISTRY OF CHINESE PRESERVED 

EGGS AND CHINESE EDIBLE 

BIRDS' NESTS 



A DISSERTATION 

SUBMITTED TO THE FACULTY 

OF THE OGDEN GRADUATE SCHOOL OF SCIENCE 

IN CANDIDACY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

DEPARTMENT OF CHEMISTRY 



BY 

CHI CHE WANG 



Private Edition, Distributed By 

THE UNIVERSITY OF CHICAGO LIBRARIES 

CHICAGO, ILLINOIS 

Reprinted from 

The Joxjrnal of Biological Chemistry, Vol. XLIX, No. 2 

December, 19 21 



Ube 'Clniversiti^ of dbtcaao 



THE CHEMISTRY OF CHINESE PRESERVED 

EGGS AND CHINESE EDIBLE 

BIRDS' NESTS 



A DISSERTATION 

SUBMITTED TO THE FACULTY 

OF THE OGDEN GRADUATE SCHOOL OF SCIENCE 

IN CANDIDACY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

DEPARTMENT OF CHEMISTRY 



BY 

CHI CHE WANG 



Private Edition, Distributed By 

THE UNIVERSITY OF CHICAGO LIBRARIES 

CHICAGO, ILLINOIS 



Reprinted from 

The Journal of Biological Chemistry, Vol. XLIX, No. 2 

December, 19 21 





J 



-<i%~^^ 



JfiAR 16 1 92 J 



Reprinted from The Journal of Biological Chemistry 
Vol. xlix, No. 2, December, 1921 



THE COMPOSITION OF CHINESE EDIBLE BIRDS' NESTS 
AND THE NATURE OF THEIR PROTEINS.* 

By CHI CHE WANG. 

{From the Nelson Morris Institute for Medical Research, Michael Reese 
Hospital, Chicago.) 

(Received for publication, September 26, 1921.) 

Appear aiice and Origin. 

The edible birds' nests are gelatinous substances produced by certain 
swifts the Collacalia, natives of Malaya (1) and Ceylon. The nests 
constructed in caves on the seashore, are collected while they are still 
moist and made into various shapes. The lowest grade is sold in the form 
of coarse powder. The higher the grade, the whiter the color and fewer 
the feathers and twigs. Owing to their high price, their use is limited to a 
delicacy at the feasts of the wealthy and a food for convalescents and the 

^^The source from which the birds make the nests has been uncertain. 
Green (2) gives three suggestions: in the alga3 found in caves where the 
swifts make their nests, fish spawn, or a secretion from the swifts them- 
selves The alga theory is disproved by the lack of vegetable cells shown 
by microscopic examination of the nests. The secretion theory is believed 
by most of the natives and has the support of Home (3) and Bernstein (4). 
The latter author found in. the birds two large salivary glands which se- 
creted much viscous mucus. The observation given in this paper shows 
that the nests consist largely of a mucin-like substance and, therefore, is 
in accord with the latter hypothesis. 

Revieio of Literature. 

The literature on the subject is limited. Descriptive statements con- 
cerning chiefly the occurrence and appearance of the nest may be found m 
Encyclopedias, Chma year books, and some semiscientific articles written 
durmg the early part of the 19th century. Green (2) and Krukenberg (5) 
are the first to give a report of a scienti fic study of the nest. Their work 

* The work reported in this article was conducted at the Nutrition 
Laboratory, Department of Home Economics, University of Chicago. It 
forms part of the thesis submitted in partial fulfillment of the require- 
ments for the degree of Doctor of Philosophy in the University of Chicago. 

429 



430 Proteins of Edible Birds' Nests 

covers the solubilities, the response to protein tests, and some observa- 
tions on hydrolysis products. Their results prove that the birds' nest 
contains both the carbohydrate and the protein radicle, belonging to the 
class of mucin-like substances, the glycoproteins. 

Scope of the Present Work. 

The present work covers the study of the general properties, 
the chemical composition, the artificial digestion, the carbohy- 
drate radicle, and the biological value of the proteins. Compari- 
son is made with other work on mucin, especially Lothrop's, 
Miiller's, and Levene's. 

The material used for this work was supplied by the Hoo Loong Edible 
Birds' Nest Store, Chicago, which imported it directly from China. It 
was of the highest grade, having somewhat the appearance of agar-agar, 
but it was extremely crisp and had tiny feathers interwoven with the 
mucilaginous material. For quantitative analysis, the material was 
ground and sifted. On sifting, most of the feathers cling together and 
may be removed, but some go through the sieve so that it is difficult to 
obtain a pure sample. 

General Properties. 

A sample boiled in distilled water for 3 hours and left there for 
several days, swells like a piece of sponge, but shows no tendency 
to dissolve. The filtrate responds to neither protein nor carbo- 
hydrate tests. 5 per cent sodium hydroxide dissolves it on 
standing 2 hours in the cold. The colorless solution responds to 
Millon's, the biuret, xanthoproteic, and Hopkins-Cole tests. The 
last reaction shows only faintly. It also has a slight reducing 
power with Fehling's reagent. A dilute acid, such as 3 per cent 
hydrochloric acid, dissolves the birds' nests only on heating. The 
solution acquires a purplish brown color, gives both protein and 
carbohydrate tests, and has a strong reducing action. 

So far the properties agree with those reported by Green (2) 
in every respect except that he found the nest insoluble in dilute 
sodium hydroxide in the cold. They also agree with the conomonly 
recognized properties of mucin. 



C. C. Wang 



431 



Chemical Analysis. 

Samples between 1.5 to 2 gm. were taken for the determination 
of moistm-e and ash. Nemnann's method given in Mathews (6) 
was used for the estimation of phosphorus, and Denis's method 
(7) for that of sulfur. An attempt was made to determine the 
ether-soluble substance, but the results were too small to be of 
significance, only 0.3 per cent. The estimation of total nitrogen 
by the Kjeldahl-Gunning method was made on samples treated 
in three different ways: (1) original birds' nest, (2) ground birds' 
nest with feathers partially removed, and (3) a sample hydro- 

TABLE I. 

Chemical Composition of Chinese Edible Birds' Nests. 







Ash. 






Total nitrogen. 










cd 




a 


No. 


Moist- 
ure. 


a> 


1 




Phos- 
phorus. 


Sulfur. 


s 


S 

-0 g 








X> 








tj 




T) a 




















<u a 






















• 




? 


.a 








"3 


•w 2 


^^ 






i 


tL 

s 


3 






a 
■3 


3 M 


So. 

"So 






.5* 


^ 










>: 0. 


>>N 






IS 


^ 


H 












w 




per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


per cent 


1 


11.88 


0.75 


1.74 


2.49 


0.035 


1.10 


8.81 


9.16 


10.57 


2 


11.41 


0.74 


1.80 


2.54 


0.034 


1.10 


8.75 


9.14 


10.24 


3 


11.52 


0.74 


1.78 


2.52 










10.33 


4 


















10.31 


Average 


11.60 


0.74 


1.77 


2.51 


0.035 


1.10 


8.78 


9.15 


10.29 



lyzed for 13^ hours in 20 per cent hydrochloric acid. Results are 
given in Table I. 

The total ash, 2.51, is almost seven times as high as that of 
submaxillary mucin, 0.37 per cent, reported by Lothrop (8). 
This high percentage of ash shows that the birds' nest is not a 
pure mucin, but more probably dried saliva. Of the total ash 
29.48 per cent is insoluble in water, but none insoluble in acid. 
Hence no sandy material is present. 

The suKur content, 1.10 per cent, is in agreement with the 
figures given by Miiller (9) for salivary mucin, 1,40 per cent, but 
it is higher than that reported by Lothrop (8), 0.55 per cent. The 



432 Proteins of Edible Birds' Nests 

discrepancy may be due to the different methods used or. in case 
of birds' nests, the presence of feathers. The phosphorus, 0.035 
per cent, is too small to be of any significance. 

The different values for nitrogen found in bu'ds' nests treated 
in three different ways may be explained by the variation in the 
feathers present. The original sample containing the most 
feathers, had the lowest figure, 8.78 per cent, while that hydrolyzed 
with the least feathers, the highest or 10.29 per cent. The ground 
and sifted sample gave 9.15 per cent. Some feathers were re- 
moved during grinding and sifting, but more of them separated 
out by clinging to the walls of the vessel on hydrolysis. They 
could then be easily removed. The percentage of nitrogen of the 
hydrolyzed material, 10.29, agrees with the value given by Miiller 
(9) for salivary mucin, 10.70 for total nitrogen, but it is lower 
than that reported by Lothrop (8), 12.49 per cent. 

Artificial Digestion. 

Artificial digestion experiments carried out in comparison with 
hard boiled egg white showed that the birds' nests were digested 
by both pepsin hydrochloric acid and trypsin though not so 
quickly as the egg. The speed of digestion was determined by 
Sorensen's titration (6). Comparison was made of the increase 
during 24 hours in the volume of 0.1 n sodium hydroxide for 
titrating 25 cc. of the peptic digest. Results were expressed in 
cc. per gm. of nitrogen in the material acted upon. For the 
birds' nests in a typical experiment this value was 9.60 cc. and 
for the egg white 15.47 cc. Similarly, the increase of 0.1 n 
hydi'ochloric acid to titrate the tryptic digest was 19.02 cc. for 
the birds' nests and 38.75 cc. for the egg white. 

The percentage of carbohydrate in the hydrolyzed birds' nests 
could not be found with accuracy. Efforts using Benedict's 
(IQ) Method 2 failed to give concordant results. The material 
was prepared in the following manner: 1 gm. of the ground and 
sifted nests was dissolved in a small amount of concentrated 
hydrochloric acid by standing over night. It was then diluted 
with distilled water to make a 5 per cent acid solution and boiled 
with a reflux condenser for 1\ hours. The hydrolyzed mixture 
was treated with phosphotungstic acid, filtered, and the filtrate 
was made up to a definite volume. 



C. C. Wang 433 

Concordant results were not obtained. Variations were there- 
fore made in the strength of acid from 3 per cent to concentrated 
and the length of hydrolysis from ^ to 8| hours. In some cases 
Levene's (11) method of introducing a little stannous chloride 
into the hydrolyzing mixture was followed. It was soon dis- 
covered that the reducing power of the birds' nest was gradually 
diminished by heating in an acid solution. A difference of 6.37 

TABLE II. 

Relation between Quantity of. Reducing Sugar and Time of Hydrolysis. 



Time. 


Reducing sugar. 


hrs. 


per cent 


i 

2 


17.36 


1 


15.99 


1 


16.03 


2 


13.63 


3 


13.11 


8§ 


10.99 



per cent of carbohydrate calculated as glucose between the samples 
hydrolyzed | and 8| hours is shown in Table II. In this experi- 
ment 20 per cent hydrochloric acid was used, the highest figure, 
in the table, 17.36 per cent, is much lower than that reported by 
Miiller (9) for the carbohydrate in salivary mucin. His is 37 per 
cent, estimated by making phenylosazone from the hydrolyzed 
mixture. 

Distribution of Nitrogen. 

For the distribution of nitrogen Van Slyke's (12) method was 
closely followed, except that the bases were precipitated from a 
volume of 250 cc. instead of 200 cc. and the correction for solu- 
bility of the basic phosphotungstates made accordingly. Approxi- 
mately 2 gm. of the ground and sifted birds' nest were taken for 
each of the four series of experiments. Several months elapsed 
between each one of the series. 

By a study of Table III it will be seen that it was possible to 
obtain a fairly complete result on the distribution of nitrogen, 
the results for the different nitrogen fractions in the three finished 
series totalling 99.84, 100.61, and 100.54 per cent, respectively. 



434 



Proteins of Edible Birds' Nests 



Series II and III are in close agreement with each other and 
Series IV slightly different, probably due to the fact that the 
material used in Series IV was purchased at a different time from 
the others. In Series II and III with the exception of cystine 
nitrogen, humin nitrogen, and the non-amino nitrogen of mono- 
amino-acids, the differences between duplicate series are all 



TABLE III. 
Distribution of Nitrogen in the Edible Birds' Nests. 



Series 


1 




TT 


in 


IV 


Average of 








II and III. 


Nitrogen. 


a 

s 


a 

2 

'3 

3 


§ 
'on 

(5 


a 
'a 

3 

o 


1 
a 

S • 


a 

a 
1 

i 


Q 

a 

3 


1 


1 

s 


1 

o 




per 
cent 


per 
cent 


per 
cent 


per 
cent 


per 
cent 


per cent 


per 
cent 


per cent 


per 

cent 


per cent 


Amide 


1.07 


10.15 1.03 


10.06 


1.05 


10.10 


1.09 


10.54 


1.04 


10.08 


Humin 


0.71 


6.75 0.65 


6.38 


0.72 


6.98 


0.70 


6.77 


0.69 


6.68 


Arginine 


1.45 


13.69 1.43 


13.96 


1.44 


13.93 


1.25 


12.12 


1.44 


13.95 


Cystine 


0.37 


3.51 0.29 


2.79 


0.41 


4.00 


0.49 


4.77 


0.35 


3.39 


Histidine 




0.60 


5.90 


0.67 


6.53 


0.67 


6.47 


0.64 


6.22 


Lysine 




0.26 


2.55 


0.25 


2.37 


0.19 


1.84 


0.26 


2.46 


Amino nitro- 




















gen of mono- 




















amino-acids. 




5.08 


49.61 


5.24 


50.76 


4.99 


48.41 


5.16 


50.19 


Non-amino ni- 




















trogen of 




















monoammo- 




















acids 






0.87 


8.54 


0.61 


5.90 


1.00 


9.62 


0.74 


7,22 


Total nitrogen 






















recovered. . . 






10.21 


99.84 


10.40 


100.61 


10.38 


100.54 


10.31 


100.23 


Total nitrogen 




















determined. 


10.57 


10.24 




10.33 




10.31 




10.29 





within the maximun experimental error allowed by Van Slyke 
(12) for pure proteins. The presence of considerable fine feathers 
with their high sulfur content and the resulting difficulty in ob- 
taining a pure sample may be the cause of the variation in the 
cystine nitrogen. The differences in the humin nitrogen are 
probably due to the carbohydrate in the nest proteins. 



C. C. Wang 435 

The humin nitrogen is shown much higher than that of any of 
the pure proteins. The highest Van Slyke (12) has reported is 3.6 
per cent for ox hemoglobin, a difference of 3.08 per cent from that 
of the nest. This high humin nitrogen may be explained as the 
result of the presence of a carbohydrate radicle in the nest pro- 
teins. Gortner and Blish (13), Gortner (14), and Hart and Sure 
(15) reported that the presence of dextrose or any other carbo- 
hydrate caused an increase of humin nitrogen during the hydrolysis 
of zein, fibrin, and casein. 

The influence of the presence of a carbohydrate radicle on the 
distribution of nitrogen is also shown by the comparatively low 
Ij^sine nitrogen and the high histidine nitrogen of the nest pro- 
teins. Thus, in case of pure casein reported by Hart and Sure 
(15) the value given for lysine nitrogen is 9.41 per cent and that 
for histidine nitrogen is 5.95 per cent. On the addition of dextrose, 
sucrose, and starch, respectively, during the hydrolysis of casein 
the corresponding values for lysine nitrogen are 7.01, 6.38, and 
5.54 per cent, and those for histidine nitrogen are 7.31, 7.65, and 
7.30 per cent. In every case, therefore, there is a decrease in the 
lysine nitrogen and an increase in the histidine nitrogen. Although 
the nature of the carbohydrate radicle in the nest has not yet 
been completely determined, it seems to possess in common with 
other carbohydrates the power of causing a redistribution of amino- 
acids on hydrolysis of protein. The presence of feathers in the 
material is undoubtedly the cause of the high value, 3.39 per cent 
for the cystine nitrogen in the nest. The highest value Van Slyke 
gave for cystine nitrogen in pure proteins is 1.25 per cent in gliadin. 

Biological Value of the Birds' Nests Proteins. 

Feeding experiments were conducted on rats at Dr. McCollum's 
laboratory,^ the School of Hygiene and Public Health, Johns 
Hopkins University, and the birds' nest was used to supplement 
a ration adequate in all respects but the character of the protein. 
Two unsatisfactory proteins, maize kernel and rolled oats, were 
chosen because they are of different character (16). Although 

1 For this part of the work I am indebted to Dr. McCollum and Miss 
Simmonds who kindly continued the experiments that I had started, and 
thus enabled me to secure the results. 



436 



Proteins of Edible Birds' Nests 



when each is fed as the sole source of protein, they have approxi- 
mately the same biological values for the support of growth, 
they are not at all similarly constituted. Oat proteins are sup- 
plemented well by the amino-acid mixture which comes from the 
digestion of gelatin, whereas the proteins of the maize kernel 
are not so supplemented in a degree which can be demonstrated 
by growth experiments with young animals. 

The complexes which form the limiting factor in those two 
proteins are different, and, therefore, if the birds' nest protein 
has a high nutritive value it should at least supplement one of 
them. Since its addition failed to supplement either protein, 
it seems very probable that the birds' nest protein is of an in- 
ferior quality. It is of course possible that when taken together 



a 


japt 


I L 


ot6 


51 
















atio 


n 




71 "^ 












Dex 


trm 






18.0 












Sal 


ts 


irf-> 




3.7 










Bu 


ttei 


fir 
'fat 


y 




5.0 










' 




/ 










wcnhi^ 








N^/ 








_ 











/ 


/ 


y 


K 


y 


^ 




"■ 




— 


l^ 


^ 


^ 


> 


/ 













Gm. 
200 
160 
120 
80 
40 



with certain other foods it might have value, but its value is 
problematical in any case, and it is certain that it is far from being 
a complete food protein. 

Lot 651 (Chart I) represents the growth curves for a group of rats which 
were restricted to a diet contauiing about 7 per cent of corn protein. The 
diet was otherwise fairly satisfactorily constituted. It failed to induce 
growth at a rate corresponding to about half the normal rate, and the 
animals became stunted when they were about half the normal adult size. 

This diet is greatly improved by the addition of even so small an amount 
of purified protein as 3 per cent of lactalbumin. This is shown in the 
growth curves of Lot 786 (Chart II). 

Lot 2071 (Chart II) shows the very slow rate of growth of a group of 
rats which were restricted to a diet comparable in all respects to those 
just mentioned except that the corn protein was fed at the plane of 6 per 
cent of the food mixture. This ration, when supplemented with a protein 



C. C. Wang 



437 



having an appreciable biological value, would become capable of inducing 
growth, as illustrated in the case of Lot 786. 

When a group of young rats were fed this diet for a period of 13 weeks 
and had been able to increase in weight but very little, and had finally 
completely failed to grow, 16 per cent of the Chinese edible birds' nest, 




equivalent to 6 per cent of N X 6.25, was added in the place of a portion 
of the carbohydrate of the diet. If the birds' nests protein had any ap- 
preciable nutritive value, it should have enabled the young animals to 
respond with growth. A wide experience has demonstrated that such 
response will be observed when the protein added is of such a nature as to 



438 Proteins of Edible Birds' Nests 

furnish certain essential amino-acids ■^hich form the limiting factor in the 
proteins of the corn (maize). In this experiment there was no growth 
following this addition of birds' nests. The only conclusion which can be 
drawn is that the birds' nests do not supplement the protein and the corn 
kernel. 

Lot 2073 (Chart III) shows that young rats cannot grow much when the 
diet contains only the amount of protein which will be furnished by a 
content of rolled oats equivalent to 40 per cent of the food mixture. This 
amounts to approximately 6 per cent of protein in the diet. Lot 1339 
(Chart III) shows that the same food mixture, supplemented with the 
proteins of flaxseed oil meal sufficient to furnish an additional 3 per cent of 
proteins, is sufficient for the maintenance of a rate of growth somewhat 
more rapid than half the rate at which the young rat is capable of growing. 
Animals on this diet have reached approximately the full adult size, and 
young have been produced. 

The fact that Lot 2073 in Period 2 failed to respond with growth when 
16 per cent of the edible birds' nest was added to the diet, leaves no room 
for doubt that this substance, although a protein of a peculiar character, 
does not supplement the proteins of the oat kernel in any appreciable 
degree. 

SUMMARY. 

1. The Chinese edible birds' nest has the properties of a pro- 
tein as well as those of a carbohydrate. It belongs, therefore, to 
the class of glycoprotein. 

2. Its percentage composition resembles that of salivary mucin. 
Its ash is high, but there is no sandy material present. It contains 
10.29 per cent nitrogen and at least 17.36 per cent carbohydrate. 

3. Artificial digestion experiments indicated that the birds' 
nest was digested by both pepsin hydrochloric acid and trypsin 
at a slower speed than boiled egg. 

4. The distribution of nitrogen showed a higher value for both 
humin nitrogen and cystine nitrogen than for pure proteins. 
The former is probably due to the carbohydrate radicle in the 
nest while the latter is due to the presence of fine feathers. Other 
fractions were similar to those of pure proteins. 

5. Feeding experiments indicate that the nest protein is prob- 
ably of an inferior qualitj'. It failed to supplement a ration ade- 
quate in all respects, except that the source of protein was de- 
rived from either maize kernel or rolled oats. Although both of 
them were unsatisfactory proteins they were different in character. 



C. C. Wans; 439 



■^to 



BIBLIOGRAPHY. 



1. Ball, J. D., Things Chinese, 3rd edition, 1900. 

2. Green, J. R., J. Physiol, 1885, vi, 40. 

3. Home, E., Phil. Tr. Roy. Soc. London, 1817, 337, quoted in Green, J. R., 

J. Physiol, 1885, vi, 40. 

4. Bernstein, J. Ornithol, 1859, 111, quoted in Green, J. R., J. Physiol, 

1885, vi, 40. 

5. Krukenberg, C. F. W., Z. Biol, 1886, xxii, 261. 

6. Mathews, A. P., Physiological chemistry. New York, 2nd edition, 1916. 

7. Denis, W., J. Biol. C/iem.,, 1910-11, viii, 401. 

8. Lothrop, A. P., J. Allied Dental Soc, 1912, vii, 410. 

9. Muller,.F., Z. Biol, 1901, xlii, 468. 

10. Benedict, S. R., /. Am. Med. Assn., 1911, Ivii, 1193. 

11. Levene, P. A., J. Biol. Chem., 1916, xxvi, 143. 

12. Van Slyke, D. D., /. Biol Chem., 1911-12, x, 15. 

13. Gortner, R. A., and Blish, M. J., /. Am. Chem. Soc, 1915, xxxvii, 1630. 

14. Gortner, R. A., /. Biol Chem., 1916, xxvi, 177. 

15. Hart, E. B., and Sure, B., J. Biol Chem., 1916, xxviii, 241. 

16. McCollum, E. V., Simmonds, N., and Pitz, W., /. Biol. Chem., 1916-17, 

xxviii, 483. 



Reprinted from The Journal op Biological Chemistrt 
Vol. xlLx, No. 2, December, 1921 



THE ISOLATION AND THE NATURE OF THE AMINO 
SUGAR OF CHINESE EDIBLE BIRDS' NESTS.* 

By CHI CHE WANG. 

{From the Nelson Morris Institute for Medical Research, Michael Reese 
Hospital, Chicago.) 

(Received for publication, September 26, 1921.) 

Literature on Carbohydrate Radicle of Glycoproteins. 

Although many attempts have been made by different investigators to 
isolate the carbohydrate group from various glycoproteins, only two 
amino sugars have so far been isolated: chitosamine (glucosamine) and 
chondrosamine. Chitosamine (1) was first isolated from lobster shells 
and later from other substances. In some cases the hydrochloride of 
chitosamine was obtained in crystalline form while in others the base was 
estimated either from the reducing power of the hydrolysis solution or 
from the phenylosazone prepared from it. Bywaters (2) estimated the 
percentage of chitosamine in the white of eggs by the former method, and 
Osborne, Jones, and Leavenworth (3) by the latter. According to the 
latter authors the reduction method for the estimation of chitosamine is 
of but little value. Ross (4) succeeded in isolating the glycosamine hydro- 
chloride in crystalline form from Boletus edulis. Her method is similar 
to the standard one given by Fischer (5) using concentrated hydrochloric 
acid to hydrolyze the substance. She demonstrated the fact that the 
glucosamine could not be isolated by hydrolyzing the substance with 
sulfuric acid. Ostwald (6) isolated a small amount of chitosamine from 
ovomucoid by dialyzing the hydrolysis solution with distilled water and 
evaporating the dialysate. Miiller (7) made an investigation on the carbo- 
hydrate radicle of mucin from the sputum of tuberculosis patients and 
from the submaxillary saliva of dogs, and after having encountered many 
failures he succeeded in isolating a crystalline compound he called glu- 
cosamine hydrochloride. The properties of this chitosamine hydrochloride 
are somewhat different from those summarized by Levene and La Forge (8) 
for the ordinary chitosamine hydrochloride, in that Mtiller's is soluble in 
95 per cent alcohol, while the latter is almost insoluble in 80 per cent 

* The work reported in this article was conducted at the Nutrition 
Laboratory, Department of Home Economics, University of Chicago. It 
forms part of the thesis submitted in partial fulfillment of the requirements 
for the degree of Doctor of Philosophy in the University of Chicago. 

441 



442 Amino Sugar of Edible Birds' Nests 

alcohol; and that his is destroyed by standing in hydrochloric acid solu- 
tion and therefore the work has to.be carried through rapidly, while the 
latter is ordinarily prepared by hydrolyzing the substance with concen- 
trated hydrochloric acid for many hours and then evaporating it on a 
water bath. 

Levene (8, 9, 10) has made a most extensive study of the carbohydrate 
radicle of mucin from pigs' stomach, funis mucin, humor vitreous mucoid, 
cornea mucoid, and mucoids from cartilage and tendon. From the mucins 
he isolated the chitosamine while from the mucoids the chondrosamine. 
The difference between the two will be discussed later. 

Isolation of the Carbohydrate. 

In attempting to isolate the carbohydrate of the nest, the 
methods of the previous investigators were first tried, but met 
with onl}'- very moderate success or entire failure. However, 
since the experiments showed something of the properties of the 
carbohydrate in contrast with the properties of the previously 
known amino sugars, they are briefly described here. 

Fischer's (5) method of preparing chitosamine by hydrolyzing 
the substance with concentrated hydrochloric acid and then 
evaporating it on a water bath was first tried. This method met 
with entire failure, as the reducing power of the birds' nest which 
had been so strong in a dilute hydrochloric acid solution hydro- 
lyzed for a short time, was completely destroyed. 

Levene's (11) method was next followed, isolating chitosamine 
from mucins by preparing the conjugated sulfuric acid first and 
then hydrolyzing the product with 20 per cent hydrochloric acid 
and a little stannous chloride. In following this method occasion- 
ally a few crystals of various forms, among which " Kreissector" 
described by Mliller (7) was also present, were obtained; but in 
no case was the yield sufficient for any analytical study. 

The third method used was Miiller's (7) for preparing chito- 
samine from salivary mucin. About 15 gm. of the nest were 
hydrolyzed with 150 cc. of 4 per cent hydrochloric acid for 3 hours 
on a water bath. The hydrolysis mixture was treated at once 
with dialyzed iron which threw down a heavy brownish black 
precipitate. The dark brown filtrate had a strong reducing 
power, but after evaporating in vacumn desiccators only a very 
few oblong crystals were formed which were not enough for re- 
crystallization. 



C. C. Wang 443 

During the many failures some of the characteristic properties 
of the carbohydrate of the birds' nest were observed. It is un- 
stable toward alkali and acid plus heat. It is soluble in 95 per 
cent ethyl and methyl alcohol. It seems to be partially absorbed 
or destroyed by protein precipitants. It is, therefore, essential 
to hydrolyze the material with dilute hydrochloric acid only long 
enough to split off the carbohydrate radicle but not to destroy it. 
The following method of preparation was then applied and 10 
gm. of beautiful white crystals were obtained after three crystal- 
lizations from alcohol and ether. 

About 300 gm. of the birds' nest were heated with 2,100 cc. of 3 per cent 
hydrochloric acid for 5 hours until the material had completely gone into 
solution, but no black precipitate was produced. (On long heating a black 
precipitate results.) The hydrolysis solution was evaporated to dryness 
over sulfuric acid and under solid sodium hydroxide in vacuum desiccators 
at room temperature. The thick black residue was extracted with 95 
per cent alcohol and the alcoholic solution was separated by centrifuge. 
The extraction was continued until the alcoholic solution gave a little or 
no reducing power. It required from fifteen to twenty extractions, at 
least. The alcoholic solution was evaporated to a very thick dark 
brown syrup under diminished pressure (30 mm.). In another case where 
a better yield was obtained it stood for several months and evaporated 
gradually at room temperature. The syrup was then taken up with a 
large quantity (about 500 cc.) of methyl alcohol. A fairly large quan- 
tity of brownish crystals thus resulted. They were filtered off and redis- 
solved in a little boiling water. The solution was filtered again and the 
brownish filtrate was treated with about eight times its volume of abso- 
lute alcohol and then with ether until no more precipitate formed. The 
crystallization was repeated until the crystals became pure white and 
seemed to be uniform on microscopic appearance; rods grouping them- 
selves in the forms of an elaborate fern leaf. The yield was about 10 gm. 

As the properties of this product seemed to be more or less 
different from those of any of the known amino sugars, it was 
thought possible that the product might be a mixture instead of 
being a single compound. Another preparation was, therefore, 
made according to the method given above except that in dis- 
solving the brown crystals from methyl alcohol less water was 
used and instead of using both absolute alcohol and ether to bring 
about the precipitation, only the former was used. This required 
much more alcohol, at least 1,500 cc. of absolute alcohol. The 
alcoholic solution which was only cloudy at first, jdelded a large 



444 Amino Sugar of Edible Birds' Nests 

crop of about 5 gm. of beautiful precipitate on standing over 
night. It was filtered by suction and after being recrystallized 
twice, was dried over suKuric acid. There are various forms of 
crystals in this fraction, among which the " Kreissector " form 
of Mtiller predominates. 

The alcoholic filtrate was treated with ether until the solution 
became cloudy. On standing over night another crop of about 
5 gm. of pure white crystals was obtained. It was filtered and 
dried as before. The crystals of this fraction are large fluffj'' 
flakes. 

Although the largest yield of the purified amino sugar was only 
about 3 per cent, the original protein contained at least 17.36 
per cent of reducing substance as shown in the previous report. 
The small yield might be due to either the unstability of the amino 
sugar or the presence of some reducing substance other than 
amino sugar in the protein. Judging from the properties of the 
sugar, the former hypothesis is probably correct. 

Properties of the Three Sets of Cnjstals. 

The three sets of crystals are all very soluble in water and 80 
per cent alcohol, fairly soluble in 95 per cent and in methyl alco- 
hol, slightly soluble in absolute alcohol, and insoluble in ether, 
chloroform, and acetone. They all taste sweet and give a strong 
test with both Molisch's and Fehling's reagents. They fail to 
respond to any of the protein reactions. When boiled with strong 
sodium hydroxide, they give off ammonia. None of them melted 
when they were placed side by side in melting point tubes and 
heated to 250°C. in a glycerol bath. . However, the alcohol-ether 
crystals started to turn dark at 142°C., the alcohol fraction at 
170°C., and the ether fraction at 150°C. At 250°C. they all 
became almost black in color. On analysis they give the follow- 
ing percentage composition: 

I. Analytical data of the alcohol-ether crystals. 
A. Determinatioa of carbon and hydrogen. 

1. 0.1858 gm. substance: 0.2254 gm. carbon dioxide and 

0.1121 gm. water. 

2. 0.1631 gm. substance: 0.1978 gm. carbon dioxide and 

0.0980 gm. water. 



C. C. Wang 445 

B. Determination of nitrogen by combustion. 

1. 0.1860 gm. substance: 10.1 cc. nitrogen at 20°C. and 

758 mm. pressure. 

2. 0.1801 gm. substance: 10.1 cc. nitrogen at 20.5°C. and 

756 mm. pressure. 

C. Determination of chlorine. 

1. 0.1541 gm. substance required for titration of hydrochloric 

acid 13.92 cc. 0.05 n silver nitrate. 

2. 0.1597 gm. substance required for titration of hydrochloric 

acid 14.35 cc. 0.05 n silver nitrate. 

3. 0.1594 gm. substance required for titration of hydrochloric 

acid 14.41 cc. 0.05 n silver nitrate. 
Cfi Hi3 Ob NCI. Calculated. H 6.50, C 33.41, N 6.51, CI 16.45. 
Found. H 6.69, C 33.09, N 6.28, CI 15.99. 

D. Determination of the optical activity. 

1. 0.3335 gm. substance in 9.947 gm. water rotated in a 1 dm. 

tube with D-light +2.58° 7 minutes after the solution 
was made, and +2.38° 28 hours later. 

Initial. Equilibrium. 

[«]d = +79.5° [af^ = +73.4° 

2. 0.2821 gm. substance in 10.816 gm. water rotated in a 1 dm. 

tube with D-light +1.81° 10 minutes after the solu- 
tion was made, and +1.72° 20 hours later. 

Initial. Equilibrium. 

[a]'p" = +71.2° * [af^ = +67.7° 

Average of (1) and (2). 

Initial. Equilibrium, 

lajy* = +75.4° [all' = +70.6° 

II. Analytical data of the alcohol fraction. 

A. Determination of nitrogen by Kjeldahl-Gunning method. 

1. 0.3140 gm. substance required for titration of ammonia 

14.56 cc. 0.1 N sulfuric acid. 

2. 0.2947 gm. substance required for titration of ammonia 

13.42 cc. 0.1 N sulfuric acid. 

3. 0.1199 gm. substance required for titration of ammonia 

5.54 cc. 0.1 N sulfuric acid. 
Cb Hi3 Oe NCI. Calculated. N 6.51. 
Found. N 6.46. 

B. Determination of optical activity. 

1. 0.3056 gm. substance in 9.9028 gm. water rotated in a 1 dm. 

tube with D-light +2.703° 10 minutes after the solu- 
tion was made, and +2.153° 22 hours later. 

Initial. Equilibrium. 

[a]o = +87.6° [at^= +69.7° 

2. 0.2997 gm. substance in 10.0797 gm. water rotated in a 1 dm. 

tube with D-light +2.56° 15 minutes after the solution 
was made, and +2.12° 20 hours later. 

Initial. Equilibrium. 

Wo= +86.1° [«];;= +71.3° 

Average of (1) and (2). 

Initial. Equilibrium. 

W;° = +86.9° [af^ = +70.5° 



446 Amino Sugar of Edible Birds' Nests 

III. Analytical data of the ether fraction. 

A. Determination of nitrogen by Kjeldahl-Gunning method. 

1. 0,2152 gm. substance required for titration of ammonia 

9.68 cc. 0.1 N sulfuric acid. 

2. 0.2016 gm. substance required for titration of ammonia 

9.02 cc. 0.1 N sulfuric acid. 
CeHis Ob NCI. 'Calculated. N 6.51. 
Found. N 6.29. 

B. Determination of optical activity. 

1. 0.2329 gm. substance in 9.8419 gm. water rotated in a 1 dm. 

tube with D-light +1.596° 10 minutes after the solu- 
tion was made, and +1.675** 22 hours later. 

Initial. Eouilibrium. 

[«]{," = +67.5° [a\'^ = +70.8° 

2. 0.3512 gm. substance in 10.2822 gm. water rotated in a 

1 dm. tube with D-light +2.24° 10 minutes after the 
solution was made, and +2.425° 20 hours later. 

Initial. Equilibrium. 

[all' = +65.6° [all," = +71.0° 

Average of (1) and (2). 

Initial. Equilibrium 

[a]^^ = +66.6° [a]'^ = +70.9° 

The phenylosazone was prepared from the alcohol-ether crystals, 
Garard and Sherman's (12) method was followed. About 1 gm. 
of the substance was placed in a small beaker with 10.80 gm. of 
freshly distilled phenylhydrazine, 12.60 gm. of glacial acetic 
acid, and 6.85 gm. of pure sodium acetate. The volume was 
made up with distilled water to 100 cc. and the mixture heated 
on a water bath for 3 hours. Unlike the osazone from glucose 
which crystallizes out during the course of heating under the 
same conditions, the osazone from this substance remained en- 
tirely in solution until it was cooled over night. This is similar 
to a preparation from glucosamine. It was so soluble in the 60 
per cent alcohol used by Garard and Sherman that to recrystallize 
their glucosazone that solvent could not be used and Levene's 
method of recrystallization from water and pyiidine was followed. 
After two recrystallizations it gave a melting point of 214° with 
rapid heating. On standing over sulfuric acid the color darkens 
and the melting point lowers markedly — 170° in one case during a 
week's standing. Samples of glucosazone made in exactly the 
same way for comparison both from glucose and glucosamine 
gave the usual melting point of 208°, and did not change on 
standing over sulfuric acid. Three tubes, one containing the 



C. C. Wang 447 

unknown osazone, the second glucosazone, and the thh'd a mix- 
ture of the two, were heated in one bath. The unknown always 
melted at a temperature about 6° above the others, while the 
mixture was either about the same or a little below the glucosazone. 
An attempt was made to study the optical activity of the osa- 
zone. As it did not dissolve completely in the usual amount of 
Neuberg's (13) alcohol pyridine mixture even after having stood 
for an hour with occasional shaking, the amount of solvent was 
doubled and a clear brown solution was secured after half an hour's 
standing. The color of the solution was, however, so deep that 
it was impossible to see through a 1 dm. tube. The solution was, 
therefore, diluted to one-fourth, the strength recommended by 
Neuberg and by Levene and it was found to rotate slightly to 
the left. 

DISCUSSION. 

An examination of the properties and the analytical data of 
the three sets shows that they are all hexosamine hydrochlorides 
and that they differ from each other only in their optical rotation 
and the temperature at which they begin to turn dark on heating. 
The latter difference might be explained on the basis of their 
purity. The one which is probably the purest, the alcohol frac- 
tion, turns dark at the highest temperature, 170°C., while the 
alcohol-ether crystals which begin to turn dark at 142°C., might 
be the least pure of the three. The degree of purity of the three 
sets of crystals is also shown by the slight difference in their 
nitrogen content. The alcohol fraction has a value of 6.46 per 
cent, the ether fraction 6.29 per cent, and the alcohol-ether crys- 
tals 6.28 per cent. The calculated value for the percentage of 
nitrogen of hexosamine is 6.51. 

The difference in the optical rotation is, however, most inter- 
esting. The average of the two determinations of optical rota- 
tion of the alcohol-ether crystals is [a]Z = + 75.4° to + 70.6° 
while those of the alcohol fraction and the ether fraction are 
W; = -f 86.9° to + 70.5° and [«]„ = + 66.6° to -f 70.9°, 
respectively. It is, therefore, clear that the three sets of crys- 
tals differ only in their initial rotations. The alcohol-ether 
crystals and the alcohol fraction have a descending rotation, 
while the ether fraction has an ascending one. Furthermore, 



448 Amino Sugar of Edible Birds' Nests 

the value of the initial rotation of the alcohol-ether crystals 
lies between those of the other two fractions and is almost equal to 
the mean of the other two, which is + 76.8°. The duplicate 
determinations were made at least 6 months apart. These facts 
seem to indicate that the alcohol fraction is probably one of the 
hexosamine hydrochlorides of the a form; the ether fraction the 
^ form, and the alcohol-ether crystals a mixture of the two. 
Thus Levene (14) reported that the optical rotation of chon- 
drosamine hydrochloride of the a form was [alJ, = -f- 129.5° 
to 4- 93.8° and that of the /3 form was [a]; = + 62.7° to + 
91.1°. No report on the properties of the a and /S forms of other 
hexosamine hydrochlorides could be found in the literature. 

Unfortunately, there is not enough material for more experi- 
mental work at present and the question as to whether the above 
hypothesis is correct has to be left unanswered. 

What one of the possible hexosamines this is, has not been 
determined. A study of Tables I and II indicates that the proper- 
ties of the unknown sugar are not in close agreement with either 
of the two known hexosamine hydrochlorides; the chitosamine 
hydrochloride or the chondrosamine hydrochloride. From Mid- 
ler's (7) description of the "glucosamine" which he obtained 
from sputum mucin it seems probable that his substance was the 
same as the alcohol-ether crystals. The highest melting point 
which he gave from his osazone was only 205°, but the discrepancy 
with our 214° might be explained by the greater purity of the 
latter. 

The unknown hexosamine hydrochloride differs from chondro- 
samine in its melting point and its optical activity, but resembles 
it in its solubility in alcohol and in giving an unstable osazone. 

The most striking point of resemblance between chitosamine 
hydrochloride and the hydrochloride of the unknown sugar lies 
in their optical activity and their stability toward concentrated 
hydrochloric acid, solubility in alcohol, and in the stability and 
the character of their osazone. Thus, chitosamine hydrochloride 
is insoluble in 80 per cent alcohol, while the hydrochloride of 
the unknown sugar is very soluble in 80 per cent alcohol, and when 
pure, soluble in 95 per cent alcohol, methyl alcohol, as well as 
absolute alcohol chitosamine hydrochloride may be prepared by 
boiling with concentrated hydrochloric acid, while this sugar is 



C. C. Wang 



449 











1 






a5 


a 












ll 
W 2 


Very sol 

ble. 
Soluble. 
Slightly 

solubl 
Soluble. 


1 


O 


d 

o 






I 


j5 


0, 








P 


o 


Is 


'o 


q3 ^ 3 fl- 


3 








a 

o 

a 

a 






Very s 
ble. 

Solubl 

Slight 
solu 

Solubl 




cr 

C 


o 

■ ^ 


d 

o 




t3 


















L^ 


, 
















.13 • 




-2 














^'c^ 


"c 


oJ ^ X a 


3 














Very s 
ble. 

Solubl 

Slight 
solu 

Solubl 


c: 
a- 


c 


d 

o 

T-l 


l~ 


















^ 








^ 


ji 








"SS 






>, 












v> 






u 


X 


3 








£ 






2 






rS 




'S. 


Q> 




3 


"c 


3 


3' 






35 


c 






o- 


02 


cc 


c 




!a3 


s 


c 


5 


c 


1— 1 


m 


^ 






03 
O 






























*«* 


















i 


3 

3 




22 




3 


a: 


_aj 




CO 

O 
H 


<0 

a 

a 

ca 
.1 




5 




3 

CO 


1— 


3 c 




S"^ 
















W ,„ 


la 




aj 












^ CO 


O 




3 






(U 






H '.s 






> 




3 


















c 


3 


c 




o 


















^ 


H 




o> 


6 a 






















•jr. 


o g 




i 


3 

"c 


3 
3 




C 

OC 




o 






iX 


02 






e 


















o 


















CO 






















o 
O 




c 

o 




^ 








a 

a: 




CO 

g 

3 

bO 

3 

CO 






*-2 










(■ 




o 






0) 




3 
o 




























a 
c. 




3 










o 


.+^ 








.1^ 










"S t 


a 




c 


^ 


es 










.s t 

.^1 


oj aj 


c 


c 


v 










1^ -^^ — 
u ^ > 
oj o -c 

U ft M -*- 


> 


"c 

bi 


u 

3 
D U 










F 


^:5 ^ 

05 <! f^ 


3 

cS 




s 










o 

CO 










§ 





450 Amino Sugar of Edible Birds' Nests 











o 


o 


o 
















o 


«o 


OS 
















d 


d 


d 
















t> 


t* 


t^ 
















+ 


+ 


+ 














_Q 


o 


o 


o 














a 


+s 


-ij 


-<-3 










. 






o 


o 


o 










u 






'^ 


OS 


O 

















ua 


CD 


O 












h 




i^ 


00 


CD 










a 


O 




+ 


+ 


+ 










^ 


3 


















o 

a 


















^ 






^ 














a 






a; 


v23 


c 








«^ 






o 


o 


o 


« 








:s 






PL, 


o 


o 










5^ 








^ 


^ 


W 








o 


























o 

CO 












-« 








I--. 












Si 




>. 




+ 












tl3 




e3 


Q 


o 












<a 




C 


«_ 


-IJ 












.e 




-3 


o 
















o 




O 

o 












e 








rH 












a 


'S' 






+ 












H 


S 


















<^ 


















tq 











o 

CO 


o 


o 

OS 


o 




«^ 


o 






1— ( 


d 


<m' 


»-H 


i-^ 




o 








t^ 


!>. 


l> 


t^ 


t>. 




g 








+ 


+ 


+ 


+ 


+ 




•c» 


•3 




^ 


o 


o 


O 


o 


o 




e 


a 




_« 


"(J 


-<o 


•i-> 


-tJ 


-tJ 




05 


03 


a) 




o 
00 


o 
>— 1 

d 


00 

CO 


o 

i6 




OS 




f-^ 


O 


a 




00 


OS 


<35 


OS 


00 




C3 


]S 


5; 




+ 


+ 


+ 


+ 


+ 




•!S> 


O 


^ 
















"S, 










1 








o 












i 


i 


i 




'^ 








1 

3 




J3 


a 


a 










a 
"3) 


a 


« .s 


3 . 


a ■ 






o 








03 _; 


03 o 


?^ ^ 


(U 'O 


a. -2 
p ° 




00 






O 


•a .a 


o a 


p o 

> 


So 
o " 
O 










































o 
















o 


o 








Q 






CO 

OS 

+ 

o 




o 

1-1 

at 

+ 


+ 

o 


OS 
+ 
o 




oi 




«, 






o 


+3 


-»J 




a 






p 




a 


-»j 


o 


o 




a 






n 


OS 


o 


CO 


• 00 




Id 






<£ 


<M 


Q 


o 


»o 


CO 






6 

1 




**H 


T-( 


V-l 


CO 


CO 


»o 




o 

C 

o 




8 


+ 


<a 


+ 


+ 


+ 




►3 


















J3 




















o 














1 
3 


1 
3 








a 

o 




o 




'■3 

1 


a 

o « 


a 

03 TJ 
|§ 
02 



C. C. Wang 451 

completely destroyed. Glucosazone melts at 208° and the osa- 
zone from this sugar at 214°. The former is more soluble in 
Neuberg's alcohol-pyridine mixture. It is stable on standing over 
sulfuric acid. The unknown osazone is unstable, darkening in 
color and lowering its melting point. 

Which amino-hexose this sugar is is therefore still undetermined. 

Levene (10) has recently made a grouping of his glycoproteins 
and their derivatives which throws light on the question of the 
birds' nest glycoproteins from tendons, cartilage, aorta, and sclera 
mucoids. He has isolated the conjugated sulfuric acid which he 
calls chondroitin sulfuric acid and which gives chondrosamine on 
hydrolysis, and from gastric mucosa, vitreous humor, funis mucin, 
cornea mucoids, and serum mucoids he has isolated mucoitin 
sulfuric acid from which chitosamine (glucosamine) is derived. 
Chondroitin sulfuric acid is highly resistant to hydrolytic agents, 
and mucoitin sulfuric acid is very easily hydrolyzed, and even 
amino sugar is often destroyed completely by the hydrolytic 
agent. It is plain from the work described in this paper that 
birds' nest glycoprotein belongs to the second group. 

He also found that different members of the second group 
behave differently, especially in the properties of their conjugated 
sulfuric acid so that he distinguishes two subdivisions A and B. 
The precursor of the unknown sugar of birds' nest is more like 
subgroup B than A. Levene's subgroup A gives a fairly insoluble 
barium salt of the conjugated suKuric acid and the subgroup B 
(the description of which was published after the work in this 
paper was done) a distinctly soluble one. The efforts of the 
writer to isolate a conjugated sulfuric acid from the birds' nest 
by the Levene early method met with almost complete failure, 
the substance behaving in the manner later described by Levene 
for subgroup B. It is, therefore, certain that the precursor of 
the unknown sugar belongs to the subgroup B of the second 
group of glycoproteins described by Levene. 

SUMMARY. 

1. A method for the solution of the amino sugar from Chinese 
edible birds' nest is described. 

2. Three sets of pure white crystals having the percentage 
composition of a hexosamine, were obtained. They are similar 



452 Amino Sugar of Edible Birds' Nests 

in all their properties except optical rotation which for two of 
them decreases on standing over night and of the third increases. 
The probable conclusion is that one of the two sets showing a 
decreasing of the optical rotation on standing is the a form of a 
hexosamine, the one which has a rise of the rotation the ^ form, 
and the third set a mixture of the two. 

3. Levene's recent description of the different groups of gly- 
coproteins and their derivatives shows that there is a striking 
resemblance between the properties of the precursor of this un- 
known sugar and those of his subgroup B of the second group. 

In conclusion I wish to express my obligation to Dr. Katharine 
Blunt, in whose laboratory the work reported in the last two 
papers was carried out, for her helpful suggestions and constant 
interest in the investigation. 

BIBLIOGRAPHY. 

1. Abderhalden, E., Handbuch der biochemischen Arbeitsmethoden, 
Berlin, 1912, vi, 76. 

2. Bywaters, H. W., J. Physiol., 1913, xlvi, p. xxxv. 

3. Osborne, T. B., Jones, D. B., and Leavenworth, C. S., Am. J. Physiol., 

1909, xxiv, 252. 

4. Eoss, W., Biochem. J., 1915, ix, 313. 

5. Fischer, E., Anleitung zur Darstellung organischer Praparate, Braun- 

schweig, 8th edition, 1908. 

6. Ostwald, A., Z. physiol. Chem., 1910, Ixviii, 173. 

7. Miiller, F., Z. Biol, 1901, xlii, 468. 

8. Levene, P. A., and La Forge, F. B., J. Biol. Chem., 1914, xviii, 123. 

9. Levene, P. A., and L6pez-Sudrez, J., /. Biol. Chem.., 1916, xxv, 511; 

xxvi, 373. Levene, P. A., and La Forge, F. B., /. Biol. Chem., 1913, 
XV, 69, 155; 1914, xviii, 238. 

10. Levene, P. A., and L<5pez-Sudrez, J., J. Biol. Chem., 1918, xxxvi, 105. 

11. Levene, P. A., and L<5pez-Su^rez, J., J. Biol. Chem., 1916, xxvi, 373; 

xxv, 514. 

12. Garard, I. D., and Sherman, H. C, /. Am. Chem. Sac, 1918, xl, 955. 

13. Neuberg, C, Ber. chem. Ges., 1898, xxxii, 3384. Neuberg, C, and 

Schewket, O., Biochem. Z., 1912, xliv, 481. 

14. Levene, P. A., /. Biol. Chem., 1917, xxxi, 609. 



LIBRARY OF CONGRESS 




014 338 893 1 « 



N. 



